Establishment of a negative-positive threshold optical density value for the enzyme-linked immunosorbent assay (ELISA) to detect soluble antigen of Renibacterium salmoninarum in Alaskan Pacific salmon

Abstract

Kldney tissues from 5231 chinook salmon Oncorhynchus tshawytscha and 3793 coho salmon 0. kisutch adults used for spawnlng were examined for soluble antigen of Renibacterium salmo-11inarum (Rs) by the enzyme-linked lmmunosorbent assay (ELISA). The purpose of this study was to develop an extens~ve data base for establishing a negative-positive threshold optical density value for Rs-negative and-positive fish uslng commercially available ELISA reagents. Statistical evaluation of the estimated distribution of Rs-negative optical density values from coho a n d chinook salmon indicated the preliminary estimated negative-positive threshold value of 0.1 was not conservative enough. i.e. there was an unacceptably high probability that a large number of low-level Rs-positive fish were not identified. Consequently, a more conservative threshold value of 0.095 was chosen that erred in ~dentifying an acceptably low number of negative fish a s positive. At this threshold optical density value the ELISA could detect about 20 ng of Rs antigen ml-l of kidney homogenate.

Bacterial species other than Renibacterium salmoninarum cross-react with antisera against R. salmoninarum but are negative for the p57 gene of R. salmoninarum as detected by the polymerase chain reaction (PCR)

“…In contrast, no fluorescence was observed in the Corynebacterium aquaticum and Carnobacterium pisclcola strains in the FAT. The kidney homogenates from the Robertson Creek female coho salmon also yielded a positive result in the ELISA: the optical density (OD) values observed were higher than the positive threshold established by Meyers et al (1993) for salmonid kidney tissues (Brown et al 1994). Similarly, the ELISA performed on the P. maltophila cells subcultured on TSA and suspended in saline also yielded positive OD values ( Table 2).…”

Comparison of the enzyme-linked immunosorbent assay (ELISA) and the fluorescent antibody test (FAT) for measuring the prevalences and levels of Renibacterium salmoninarum in wild and hatchery stocks of salmonid fishes in Alaska. USA

“…Statistical analyses of ELISA data are described in Meyers et al (1993) including the correction statistic for interassay comparison of data by . This statistic was used to correct for any minor fluctuations in optical density values of test fish samples d u e to new reagents or day-to-day changes in equipment performance.…”

Interpreting dual ELISA and qPCR data for bacterial kidney disease of salmonids

“…The 96-well format permits processing of large sample numbers, and commercially available reagents facilitate inter-laboratory calibration. The procedure is robust because sample preparation is simple, the target antigens are stable and abundant, and the assay has a sensitivity of approximately 20 to 40 ng ml -1 of homogenate (Meyers et al 1993). Renibacterium salmoninarum secretes abundant quantities of major soluble antigen (MSA), which is the dominant ELISA target (Pascho & Mulcahy 1987), and tissue accumulations of MSA can persist for up to 2 mo (Pascho et al 1997).…”